Dec 8, 1987 - JOHN R OLIVER, NICHOLAS TWIDALE, CAROL LAKIN, MICHAEL CAIN,. ANDREW M TONKIN. From the Department of Medicine, Flinders ...
Br Heart J 1988;59:458-62
Plasma atrial natriuretic polypeptide concentrations during and after reversion of paroxysmal supraventricular tachycardias JOHN R OLIVER, NICHOLAS TWIDALE, CAROL LAKIN, MICHAEL CAIN, ANDREW M TONKIN From the Department of Medicine, Flinders Medical Centre, South Australia
Plasma concentrations of immunoreactive atrial natriuretic polypeptide were raised in 22 of 23 patients with paroxysmal supraventricular tachycardia and in all seven patients with atrial flutter. Plasma concentrations of atrial natriuretic polypeptide rose soon after the onset of supraventricular tachycardia. A sample taken 30 minutes after reversion to sinus rhythm (pharmacological or non-pharmacological) showed a significant fall in 19 of the 23 patients with paroxysmal supraventricular tachycardia and all seven patients with atrial flutter. Because atrial natriuretic polypeptide has powerful natriuretic and diuretic properties, an increase may contribute considerably to the polyuria that is often associated with episodes of SUMMARY
supraventricular tachycardia. Mammalian atrial tissue has been shown to contain secretory-like storage granules with characteristics similar to those of endocrine peptide secretory cells."2 These granules were shown to contain a polypeptide hormone, which is first synthesised as a 151 amino acid prohormone.34 The prohormone is further processed to several smaller peptides, one of which is a natriuretic polypeptide, a 28 amino acid polypeptide with potent natriuretic, diuretic, and vasorelaxant properties.' The physiological role of atrial natriuretic polypeptide, or indeed any of the post-translational products, is unknown; however, there is a considerable body ofevidence to suggest that this peptide may help to maintain sodium ion and water balance and modulate the renin-angiotensin aldosterone system.8 A major stimulus for the release of atrial natriuretic polypeptide into blood seems to be atrial distension produced by volume loading or increased dietary intake of sodium ion.9 10 Paroxysmal supraventricular tachycardias are often associated with considerable polyuria and reports of raised concentrations of plasma atrial natriuretic polypeptide in such patients suggest that
this substance may account for the combined diuresis and natriuresis. 11-13 We measured plasma concentrations of atrial natriuretic polypeptide before and after reversion to sinus rhythm in patients with paroxysmal supraventricular tachycardia and those with atrial fibrillation or flutter. Patients and methods PATIENTS
This study was carried out within the guidelines set down by the National Health and Medical Research Council of Australia and was approved by the institute's human ethics advisory committee. We studied twenty three patients admitted to our casualty department with paroxysmal supraventricular tachycardia (18 women and 5 men, aged 1781 (mean 53) years). Diagnosis was confirmed by 12 lead electrocardiography which showed a regular, narrow QRS tachycardia in all 23 patients. Patients with acute or chronic congestive cardiac failure were specifically excluded. In addition, we studied seven patients (five men and two women (mean age 60) with atrial fibrillation or flutter and five control patients (three men and two Requests for reprints to Dr John R Oliver, Department of Medicine, women (mean age 50)) without arrhythmias who had Flinders Medical Centre, Bedford Park 5042, South Australia, had intravenous lines inserted in the casualty departAustralia. ment for other indications. Renal function was assessed in all patients. Plasma Accepted for publication 8 December 1987 458
Atrial natriuretic polypeptide concentrations in supraventricular tachycardia 459 creatinine concentrations were within the normal Specificity and characterisation of antiserum. range (O06-0-12 mmol/l) in all but two patients (0-14 Antiserum to atrial natriuretic polypeptide 1-28 and 0 21 mmol/l). was raised in sheep after conjugation of synthetic Fifteen patients were receiving concomitant human cx atrial natriuretic polypeptide 1-28 to medication when they were studied. This included a bovine thyroglobulin (Sigma Chemical USA) with # blocker in one, j blocker plus digoxin in two, ethylcarbodiimide (Pierce Chemicals, USA). The digoxin alone in one, quinidine in two, verapamil in antiserum cross reacted to an equal degree with one, diuretic agents in three, theophylline in one, atriopeptins I, II, III, but it did not cross react with thyroxine in two, and non-steroidal anti- cardiodilatin 1-16, angiotensin II, vasopressin, inflammatory drugs in two. oxytocin, neuropeptide Y, somatostatin, ( endorA first blood sample was taken after insertion of a phin, leu and met enkephalins and dynorphins 1-8 peripheral intravenous line and before attempted and 1-13. reversion and a second sample was taken 30 minutes We used the MK model extended least squares after reversion to sinus rhythm. non-linear curve fitting program to analyse antibody binding affinity. Two binding sites were found: the BLOOD COLLECTION AND RADIOIMMUNOASSAY first had a dissociation constant (Kd) of 37-58 pmol/l OF ATRIAL NATRIURETIC POLYPEPTIDE IN and affinity constant (Ka) of 2-7 x 10 l/pmol and the PLASMA second a Kd of 122-5 pmol/l and Ka of 0-08 x Blood collection and extraction 10 l/pmol. Venous blood (10 ml) was collected into a chilled Radioimmunoassaywas carried out in 10 x 75 mm tube containing edetic acid and aprotinin (10 000 units; Bayer, Australia). We took care to avoid borosilicate glass tubes under non-equilibrium haemolysis, which causes loss of immunoreactive conditions at 4°C. Duplicate volumes (0-20 ml) ofthe atrial natriuretic polypeptide in plasma. Plasma was plasma extract were incubated with 100 p1 of sheep separated immediately by centrifugation, snap antiserum to human x atrial natriuretic polypeptide (final concentration 1:6000), followed by a further frozen, and stored at - 20°C until assay. two day incubation, after the addition of 100 p1 of (3[1251] iodotyrosyl 28) a atrial natriuretic polypeptide Extraction of plasma Plasma (1 ml) was extracted ovemight at 4°C with (5000 counts/min equivalent to 1-2 pg tracer mass) 1-8 ml acidified ethanol (95: 5 parts of 95%O ethanol: (Amersham, Australia). Synthetic human a atrial 6 mol/l acetic acid) followed by centrifugation, and natriuretic polypeptide was used to generate a stanlyophilisation of the supernatant. The dried extract, dard curve over the range 2-1000 pg/tube. Bound and free atrial natriuretic polypeptide were equivalent to 1 0 ml of plasma, was reconstituted with 0 50 ml of assay buffer (radioimmunoassay separated by the addition of 100 p1 of carrier sheep section) and assayed for atrial natriuretic polypep- serum (diluted 1:250) and 100 pl of donkey antisheep immunoglobulin serum (diluted 1:5) followed by tide. incubation at room temperature for three hours and was extraction of the procedure efficiency The determined for each patient's sample by the addition centrifugation. The pellet was counted in a NEN of radiolabelled human a atrial natriuretic polypep- 1600 gamma counter (Nuclear Enterprises). tide to a separate 1 ml volume of patient's plasma. Standards were set up in triplicate in the presence of This plasma was extracted as described above. After 0-20 ml of an extract of charcoal stripped plasma reconstitution in assay buffer (0 50 ml) a 200 p1 (hormone free) so as to reproduce the assay condivolume was taken and its radioactive content tions used for the patient plasma samples. Concenmeasured and recovery was estimated. Recovery of tration of plasma atrial natriuretic polypeptide was radiolabelled human a atrial natriuretic polypeptide determined from the standard curve, plotted as the from plasma samples from both the controls and the logit-log of the ratio of bound counts of unknown to bound counts of the zero standard versus concentrapatients was 78(6)% (mean (SEM)). tion (pg/tube). The standard curve was linear over the range 10 to 600 pg/tube and the sensitivity of the Radioimmunoassay of atrial natriuretic polypeptide Assay buffer.-For all procedures in the assay we assay was 2 pg/tube (2 from the mean ± SD of the used 0-05 mol/l sodium phosphate buffered saline zero standard). The intra and inter assay variability pH 7-4 containing aprotinin (250 U/ml), edetic acid were 8 and 150% respectively. Characterisation of plasma atrial natriuretic (0 01 mol/l), 0-25 g % bovine serum albumin (Sigma Chemical, radioimmunoassay grade), and polypeptide measured by radioimmunoassay.-Plasma thiomerosal (0-6 mmol) (Sigma Chemical, as a samples collected before and after reversion were pooled separately, extracted as above, and the dried preservative).
460 extracts were analysed by high pressure liquid chromatography on a C18 reverse phase column and with an acetonitrile linear gradient elution profile. Analysis of the column eluant showed one major peak of immunoreactivity for both the plasma samples that coincided with the elution profile of the synthetic human a atrial natriuretic polypeptide (results not shown). The plasma extracts were further characterised by serial dilution experiments. Pooled extracts of plasma obtained before and after reversion diluted in parallel with the synthetic human a atrial natriuretic polypeptide standard (results not shown). Concentration of atrial natriuretic polypeptide in controls.-Atrial natriuretic polypeptide was measured in blood samples from 34 normotensive healthy volunteers (14 men, 20 women) with normal dietary intake of sodium and electrolyte status which were treated and extracted as described above. Subjects remained seated for 15 minutes before a blood sample was taken between 9 am and 10.30 am. The upper and lower limits of the normal range were 70 pg/ml and 4 pg/ml respectively and no sex dependent differences were found (men 36 (14) pg/ ml, women 33 (16) pg/ml) (mean (SEM)). I
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Oliver, Twidale, Lakin, Cain, Tonkin
Results Figure 1 shows plasma concentrations of atrial natriuretic polypeptide before and after reversion to sinus rhythm in the 23 patients with paroxysmal supraventricular tachycardia. During paroxysmal supraventricular tachycardia 22 of 23 patients had atrial natriuretic polypeptide concentrations above the upper limit for normal subjects in this laboratory (70 pg/ml). In one patient plasma atrial natriuretic polypeptide was within the normal range and did not change after reversion. Paroxysmal supraventricular tachycardia was converted to sinus rhythm by intravenous verapamil (1-20 mg) in 16 patients, intravenous digoxin (250-500 jug) in one, vagal manoeuvres in three, or a combination of these in three. In 19 patients concentrations of plasma atrial natriuretic polypeptide fell 30 minutes after reversion, in five to within the normal range. The mean concentration of plasma atrial natriuretic polypeptide was 256 (43) pg/ml before reversion and 120 (19) mg/ml 30 minutes after reversion (2 p < 0.005 by paired Student's t test). The fall in the concentration of atrial natriuretic polypeptide seemed to be independent of the mode of reversion of paroxysmal supraventricular tachycardia. There was a weak inverse relation between the rate of paroxysmal supraventricular tachycardia (140220 beats/min) and plasma concentrations of atrial natriuretic polypeptide (r = -0A45, p = 0.017 by
400 300
n-7
300X Plusm
2010
ANP (pg/mi)
200
Plasma
ANP (pg/mi)
100100
After
120(19) Fig 1 Plasma concentration (mean(SEM)) of atrial natriuretic polypeptide (ANP) (pg/ml) before and 30 minutes after reversion in 23 patients with paroxysmal supraventricular tachycardia. The normal range is also shown.
0*
B3efore
156 (33)
2pc0.01
After
68 (22)
Fig 2 Plasma concentration (mean (SEM) ) of atrial natriuretic polypeptide (ANP) (pg/ml) before and 30 minutes after reversion in seven patients with atrial fibrillation or flutter. The normal range is also shown.
461 Atrial natriuretic polypeptide concentrations in supraventricular tachycardia Pearson's test). The duration of paroxysmal paroxysmal supraventricular tachycardia in supraventricular tachycardia (10 min-3 days) had particular) are associated with raised concentrations no apparent effect on plasma atrial natriuretic of plasma atrial natriuretic polypeptide that fall polypeptide concentrations; one patient had con- rapidly and apparently independently of the mode of siderably raised plasma atrial natriuretic polypeptide reversion whether this is pharmacological or non(84 pg/ml) with paroxysmal supraventricular pharmacological. tachycardia lasting only 10 minutes. In the seven patients with atrial fibrillation or We thank Dr R Murphy for high pressure liquid flutter, plasma concentrations of atrial natriuretic chromatography analysis. This work was carried out polypeptide were raised before reversion was with part support from the National Health and attempted (156 (33) pg/ml) and fell after reversion Medical Research Council of Australia. (68 (22) pg/ml) (fig 2).
Discussion Our results show that patients with supraventricular tachycardia, particularly paroxysmal supraventricular tachycardia, and atrial fibrillation or flutter have considerably higher plasma concentrations of atrial natriuretic polypeptide than healthy controls. These results are consistent with earlier studies of small numbers of patients and animal studies."'" Given that atrial natriuretic polypeptide has natriuretic and diuretic properties, the increase may account for the polyuria reported in patients with supraventricular tachycardias.' 202' Others have suggested that the polyuria may be caused by suppressed vasopressin release2' 22; however, such a mechanism does not account for the striking natriuresis associated with this condition.20 Plasma concentrations of atrial natriuretic polypeptide may rise early after the onset of paroxysmal supraventricular tachycardia and remain raised for prolonged periods of tachycardia, independent of clinical indices of left ventricular failure. The weak inverse relation between the rate of paroxysmal supraventricular tachycardia and plasma concentrations of atrial natriuretic polypeptide remains unexplained. One possible explanation is that the atrial cardiocyte becomes fatigued and supplies of atrial natriuretic polypeptide are exhausted. This would be an unusual phenomenon in endocrine cells but would be consistent with the fall in concentrations of atrial natriuretic polypeptide during tachycardia in one of the patients with atrial flutter (119 pg/ml to 44 pg/ml) that occurred without a change in rate of tachycardia and before reversion was attempted. Although atrial pressure increases in this type of tachycardia and may act as the stimulus for secretion of atrial natriuretic polypeptide," alternative intrinsic mechanism(s) associated with the arrhythmias could also mediate release of atrial natriuretic polypeptide. Further studies will be required to elucidate the mechanism(s) responsible. In conclusion, supraventricular tachycardia (and
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